Tyrosyl tRNA synthetase polynucleotides

ABSTRACT

The invention provides tyrS polypeptides and DNA (RNA) encoding tyrS polypetides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing tyrS polypeptides to screen for antibacterial compounds.

RELATED APPLICATION

This application claims benefit of UK application number 9608001.5,filed Apr. 18, 1996.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the tyrosyl tRNA synthetase family, hereinafter referredto as "tyrS".

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneunoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptoccus pneunoniae strains which are resistantto some or all of the standard antibiotics. This has created a demandfor both new anti-microbial agents and diagnostic tests for thisorganism.

The t-RNA synthetases have a primary role in protein synthesis accordingto the following scheme:

    Enzyme+ATP+AAEnzyme.AA-AMP+PPi

    Enzyme.AA-AMP+t-RNAEnzyme+AMP+AA-t-RNA

in which AA is an amino acid.

Inhibition of this process leads to a reduction in the levels of chargedt-RNA and this triggers a cascade of responses known as the stringentresponse, the result of which is the induction of a state of dormancy inthe organism. As such selective inhibitors of bacterial t-RNA synthetasehave potential as antibacterial agents. One example of such is mupirocinwhich is a selective inhibitor of isoleucyl t-RNA synthetase. Othert-RNA synthetases are now being examined as possible anti-bacterialtargets, this process being greatly assisted by the isolation of thesynthetase.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown Bacillus subtilis tyrosyl tRNA synthetase protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel tyrS polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2] and a known amino acidsequence or sequences of other proteins such as Bacillus subtilistyrosyl tRNA synthetase protein.

It is a further object of the invention to provide polynucleotides thatencode tyrS polypeptides, particularly polynucleotides that encode thepolypeptide herein designated tyrS.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding tyrS polypeptides comprisingthe sequence set out in Table 1 [SEQ ID NO:1] which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel tyrS protein from Streptoccus pneunoniae comprising the amino acidsequence of Table 1 [SEQ ID NO:2], or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Streptococcus pneurmoniae 0100993 strain contained in thedeposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding tyrS, particularly Streptococcus pneumnoniaetyrS, including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of tyrS and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStreptococcus pneumoniae referred to herein as tyrS as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of tyrS polypeptide encoded by naturally occurring alleles ofthe tyrS gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned tyrS polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing tyrSexpression, treating disease, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid, assaying genetic variation, andadministering a tyrS polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStreptococcus pneumoniae bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to tyrS polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against tyrS polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound, and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided tyrS agonists and antagonists, preferably bacteriostatic orbactericidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a tyrS polynucleotide or a tyrS polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J.Molec. Biol. 215: 403-410 (1990). The BLAST X program is publiclyavailable from NCBI and other sources (BLAST Manual, Altschul, S., etal., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol.Biol. 215: 403-410 (1990). As an illustration, by a polynucleotidehaving a nucleotide sequence having at least, for example, 95%"identity" to a reference nucleotide sequence of SEQ ID NO: 1 it isintended that the nucleotide sequence of the polynucleotide is identicalto the reference sequence except that the polynucleotide sequence mayinclude up to five point mutations per each 100 nucleotides of thereference nucleotide sequence of SEQ ID NO: 1. In other words, to obtaina polynucleotide having a nucleotide sequence at least 95% identical toa reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5' or 3'terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Analogously, by a polypeptidehaving an amino acid sequence having at least, for example, 95% identityto a reference amino acid sequence of SEQ ID NO:2 is intended that theamino acid sequence of the polypeptide is identical to the referencesequence except that the polypeptide sequence may include up to fiveamino acid alterations per each 100 amino acids of the reference aminoacid of SEQ ID NO: 2. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel tyrS polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel tyrS of Streptococcuspneumoniae, which is related by amino acid sequence homology to Bacillussubtilis tyrosyl tRNA synthetase polypeptide. The invention relatesespecially to tyrS having the nucleotide and amino acid sequences setout in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2] respectively,and to the tyrS nucleotide sequences of the DNA in the deposited strainand amino acid sequences encoded thereby.

                                      TABLE 1                                     __________________________________________________________________________    tyrS Polynucleotide and Polypeptide Sequences                                 __________________________________________________________________________    (A) Sequences from Streptococcus pneumoniae tyrS                              polynucleotide sequence [SEQ ID NO:1].                                        __________________________________________________________________________    5'                                                                            1      ATGCACATTT TTGATGAGCT AAAAGAGCGT GGTTTGATAT TTCAAACGAC                    51  TGATGAAGAA GCTTTGCGTA AAGCCCTAGA AGAAGGTCAA GTTTCTTATT                    101 ATACTGGCTA CGATCCAACT GCTGACAGCC TTCACCTAGG CCACCTTGTC                    151 GCAATCTTGA CAAGTCGTCG CTTGCAACTA GCAGGTCACA AACCTTATGC                    201 GCTCGTTGGC GGTGCTACAG GTCTCATCGG AGATCCGTCC TTCAAAGATG                    251 CTGAACGTAG TCTCCAAACA AAAGACACAG TAGATGGCTG GGTCAAGTCT                    301 ATCCAAGGAC AACTTTCTCG TTTTCTTGAC TTTGAAAATG GCGAAAACAA                    351 GGCTGTCATG GTCAACAACT ACGACTGGTT TGGCAGCATC AGCTTCATTG                    401 ACTTCCTCCG TGATATTGGA AAATACTTCA CGGTCAACTA CATGATGAGT                    451 AAGGAATCTG TTAAAAAACG GATCGAAACA GGAATTTCTT ACACTGAGTT                    501 CGCTTACCAA ATCATGCAAG GGTACGACTT CTTCGTCCTT AACCAAGACC                    551 ATAATGTCAC TCTTCAAATC GGTGGTTCTG ACCAGTGGGG AAATATGACA                    601 GCTGGTACCG AATTGCTTCG TCGTAAGGCG GACAAGACTG GTCACGTTAT                    651 CACTGTTCCA CTAATCACAG ATGCAACTGG TAAAAAATTT GGTAAATCAG                    701 AAGGAAATGC CGTCTGGCTC AATCCCGAAA AGACTTCTCC ATACGAAATG                    751 TACCAATTCT GGATGAACGT GATGGACGCT GACGCTGTTC GCTTCTTGAA                    801 AATCTTTACT TTCTTGTCAC TTGATGAGAT TGAAGATATT CGTAAACAAT                    851 TTGAAGCAGC GCCACACGAA CGCTTGGCTC AAAAAGTCTT GGCTCGTGAA                    901 GTTGTTACAC TTGTTCACGG AGAAGAAGCC TACAAAGAAG CACTTAACAT                    951 CACTGAGCAA CTCTTTGCAG GAAACATCAA AAACCTTTCT GTCAAAGAGC                    1001                                                                              TCAAACAAGG ACTTCGTGGT GTGCCAAACT ACCAAGTACA GGCAGACGAA                    1051                                                                              AACAACAATA TCGTGGAACT GCTCGTCTCA TCTGGTATAG TTAACTCAAA                    1101                                                                              ACGCCAAGCC CGTGAAGACG TCCAAAACGG AGCCATCTAC GTAAACGGCG                    1151                                                                              ACCGCATCCA AGACCTTGAC TATGTCTTGA GTGACGCTGA TAAGTTAGAA                    1201                                                                              AATGAACTGA CTGTTATCCG TCGTGGGAAG AAAAAATACT TTGTATTGAC                    1251                                                                              TTACTAA-3'                                                             __________________________________________________________________________    (B) tyrS polypeptide sequence deduced from the polynucleotide                 sequence in this table [SEQ ID NO:2].                                         __________________________________________________________________________    NH.sub.2                                                                      1      MHIFDELKER GLIFQTTDEE ALRKALEEGQ VSYYTGYDPT ADSLHLGHLV                    51  AILTSRRLQL AGHKPYALVG GATGLIGDPS FKDAERSLQT KDTVDGWVKS                    101 IQGQLSRFLD FENGENKAVM VNNYDWFGSI SFIDFLRDIG KYFTVNYMMS                    151 KESVKKRIET GISYTEFAYQ IMQGYDFFVL NQDHNVTLQI GGSDQWGNMT                    201 AGTELLRRKA DKTGHVITVP LITDATGKKF GKSEGNAVWL NPEKTSPYEM                    251 YQFWMNVMDA DAVRFLKIFT FLSLDEIEDI RKQFEAAPHE RLAQKVLARE                    301 VVTLVHGEEA YKEALNITEQ LFAGNIKNLS VKELKQGLRG VPNYQVQADE                    351 NNNIVELLVS SGIVNSKRQA REDVQNGAIY VNGDRIQDLD YVLSDADKLE                    401 NELTVIRRGK KKYFVLTY-COOH                                               __________________________________________________________________________    (C) Polynucleotide sequence embodiments [SEQ ID NO:1].                        __________________________________________________________________________    X-(R.sub.1).sub.n                                                             1      ATGCACATTT TTGATGAGCT AAAAGAGCGT GGTTTGATAT TTCAAACGAC                    51  TGATGAAGAA GCTTTGCGTA AAGCCCTAGA AGAAGGTCAA GTTTCTTATT                    101 ATACTGGCTA CGATCCAACT GCTGACAGCC TTCACCTAGG CCACCTTGTC                    151 GCAATCTTGA CAAGTCGTCG CTTGCAACTA GCAGGTCACA AACCTTATGC                    201 GCTCGTTGGC GGTGCTACAG GTCTCATCGG AGATCCGTCC TTCAAAGATG                    251 CTGAACGTAG TCTCCAAACA AAAGACACAG TAGATGGCTG GGTCAAGTCT                    301 ATCCAAGGAC AACTTTCTCG TTTTCTTGAC TTTGAAAATG GCGAAAACAA                    351 GGCTGTCATG GTCAACAACT ACGACTGGTT TGGCAGCATC AGCTTCATTG                    401 ACTTCCTCCG TGATATTGGA AAATACTTCA CGGTCAACTA CATGATGAGT                    451 AAGGAATCTG TTAAAAAACG GATCGAAACA GGAATTTCTT ACACTGAGTT                    501 CGCTTACCAA ATCATGCAAG GGTACGACTT CTTCGTCCTT AACCAAGACC                    551 ATAATGTCAC TCTTCAAATC GGTGGTTCTG ACCAGTGGGG AAATATGACA                    601 GCTGGTACCG AATTGCTTCG TCGTAAGGCG GACAAGACTG GTCACGTTAT                    651 CACTGTTCCA CTAATCACAG ATGCAACTGG TAAAAAATTT GGTAAATCAG                    701 AAGGAAATGC CGTCTGGCTC AATCCCGAAA AGACTTCTCC ATACGAAATG                    751 TACCAATTCT GGATGAACGT GATGGACGCT GACGCTGTTC GCTTCTTGAA                    801 AATCTTTACT TTCTTGTCAC TTGATGAGAT TGAAGATATT CGTAAACAAT                    851 TTGAAGCAGC GCCACACGAA CGCTTGGCTC AAAAAGTCTT GGCTCGTGAA                    901 GTTGTTACAC TTGTTCACGG AGAAGAAGCC TACAAAGAAG CACTTAACAT                    951 CACTGAGCAA CTCTTTGCAG GAAACATCAA AAACCTTTCT GTCAAAGAGC                    1001                                                                              TCAAACAAGG ACTTCGTGGT GTGCCAAACT ACCAAGTACA GGCAGACGAA                    1051                                                                              AACAACAATA TCGTGGAACT GCTCGTCTCA TCTGGTATAG TTAACTCAAA                    1101                                                                              ACGCCAAGCC CGTGAAGACG TCCAAAACGG AGCCATCTAC GTAAACGGCG                    1151                                                                              ACCGCATCCA AGACCTTGAC TATGTCTTGA GTGACGCTGA TAAGTTAGAA                    1201                                                                              AATGAACTGA CTGTTATCCG TCGTGGGAAG AAAAAATACT TTGTATTGAC                    1251                                                                              TTACTAA                                                                (R.sub.2).sub.n -Y                                                            __________________________________________________________________________    (D) Polypeptide sequence embodiments [SEQ ID NO:2].                           __________________________________________________________________________    X-(R.sub.1).sub.n                                                             1      MHIFDELKER GLIFQTTDEE ALRKALEEGQ VSYYTGYDPT ADSLHLGHLV                    51  AILTSRRLQL AGHKPYALVG GATGLIGDPS FKDAERSLQT KDTVDGWVKS                    101 IQGQLSRFLD FENGENKAVM VNNYDWFGSI SFIDFLRDIG KYFTVNYMMS                    151 KESVKKRIET GISYTEFAYQ IMQGYDFFVL NQDHNVTLQI GGSDQWGNMT                    201 AGTELLRRKA DKTGHVITVP LITDATGKKF GKSEGNAVWL NPEKTSPYEM                    251 YQFWMNVMDA DAVRFLKIFT FLSLDEIEDI RKQFEAAPHE RLAQKVLARE                    301 VVTLVHGEEA YKEALNITEQ LFAGNIKNLS VKELKQGLRG VPNYQVQADE                    351 NNNIVELLVS SGIVNSKRQA REDVQNGAIY VNGDRIQDLD YVLSDADKLE                    401 NELTVIRRGK KKYFVLTY-(R.sub.2).sub.n -Y                                 __________________________________________________________________________    (E) Sequences from Streptococcus pneumoniae tyrS                              polynucleotide ORF sequence [SEQ ID NO:3].                                    __________________________________________________________________________    5'                                                                            1      AAGAGCTTGC GTAAAGCCCT AGAAGAAGGT CAAGTTTCTT ATTATACTGG                    51  CTACGATCCA ACTGCTGACA GCCTTCACCT AGGCCACCTT GTCGCAATCT                    101 TGACAAGTCG TCGCTTGCAA CTAGCAGGTC ACAAACCTTA TGCGCTCGTT                    151 GGCGGTGCTA CAGGTCTCAT CGGAGATCCG TCCTTCAAAG ATGCTGAACG                    201 TAGTCTCCAA ACAAAAGACA CAGTAGATGG CTGGGTCAAG TCTATCCAAG                    251 GACAACTTTC TCGTTTTCTT GACTTTGAAA ATGGCGAAAA CAAGGCTGTC                    301 ATGGTCAACA ACTACGACTG GTTTGGCAGC ATCAGCTTCA TTGACTTCCT                    351 CCGTGATATT GGAAAATACT TCACGGTCAA CTACATGATG AGTAAGGAAT                    401 CTGTTAAAAA ACGGATCGAA ACAGGAATTT CTTACACTGA GTTCGCTTAC                    451 CAAATCATGC AAGGGTACGA CTTCTTCGTC CTTAACCAAG ACCATAATGT                    501 CACTCTTCAA ATCGGTGGTT CTGACCAGTG GGGAAATATG ACAG-3'                    __________________________________________________________________________    (F) tyrS polypeptide sequence deduced from the polynucleotide                 ORF sequence in this table [SEQ ID NO:4].                                     __________________________________________________________________________    NH.sub.2                                                                      1      KSLRKALEEG QVSYYTGYDP TADSLHLGHL VAILTSRRLQ LAGHKPYALV                    51  GGATGLIGDP SFKDAERSLQ TKDTVDGWVK SIQGQLSRFL DFENGENKAV                    101 MVNNYDWFGS ISFIDFLRDI GKYFTVNYMM SKESVKKRIE TGISYTEFAY                    151 QIMQGYDFFV LNQDNHVTLQ IGGSDQWGNM T-COOH                                __________________________________________________________________________

Deposited materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Apr. 11, 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit. On Apr.17, 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli wassimilarly deposited with the NCIMB and assigned deposit number 40800.The Streptococcus pneurnoniae strain deposit is referred to herein as"the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length tyrS gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1[SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of tyrS, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NOS:2 and 4] or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNOS:2 and 4], and more preferably at least 90% similarity (morepreferably at least 90% identity) to a polypeptide of Table 1 [SEQ IDNOS:2 and 4] and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to a polypeptide of Table 1 [SEQID NOS:2 and 4] and also include portions of such polypeptides with suchportion of the polypeptide generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) [SEQ ID NO:2] wherein, at the amino terminus, X is hydrogen,and at the carboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is anyamino acid residue, and n is an integer between 1 and 1000. Any stretchof amino acid residues denoted by either R group, where R is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with tyrS polypeptides fragments maybe "free-standing," or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NOS:2 and 4], orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Streptococcus pneumoniae,are also preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of tyrS, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the tyrS polypeptide havinga deduced amino acid sequence of Table 1 [SEQ ID NOS:2 and 4] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NOS:1 and 3], a polynucleotide of theinvention encoding tyrS polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Streptococcus pneumoniae0100993 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 [SEQ ID NOS:1 and 3],typically a library of clones of chromosomal DNA of Streptococcuspneumoniae 0100993 in E.coli or some other suitable host is probed witha radiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York (1989). (see in particular ScreeningBy Hybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO:1] was discovered in a DNA library derivedfrom Streptococcus pneumoniae 0100993.

The DNA sequence set out in Table 1 [ SEQ ID NOS: 1] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NOS:2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The start codon of the DNA in Table 1 isnucleotide number 1 and last codon that encodes and amino acid is number1254, the stop codon being the next codon following this last codonencoding an amino acid.

tyrS of the invention is structurally related to other proteins of thetyrosyl tRNA synthetase family, as shown by the results of sequencingthe DNA encoding tyrS of the deposited strain. The protein exhibitsgreatest homology to Bacillus subtilis tyrosyl tRNA synthetase proteinamong known proteins. tyrS of Table 1 [SEQ ID NO:2] has about 58%identity over its entire length and about 75% similarity over its entirelength with the amino acid sequence of Bacillus subtilis tyrosyl tRNAsynthetase polypeptide.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is the polynucleotide ofcomprising nucleotide 1 to 1254 set forth in SEQ ID NO:1 of Table 1which encodes the tyrS polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C)[SEQ ID NO:1] wherein, at the 5' end of the molecule, X ishydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae tyrShaving the amino acid sequence set out in Table 1 [SEQ ID NO:2]. Theterm also encompasses polynucleotides that include a single continuousregion or discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or an insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingtyrS variants, that have the amino acid sequence of tyrS polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof tyrS.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding tyrS polypeptide having an amino acid sequence set out in Table1 [SEQ ID NOS:2 and 4], and polynucleotides that are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding tyrS polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 [SEQ ID NO: 1].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO: 1 or SEQ ID NO:3 under stringent hybridization conditionswith a probe having the sequence of said polynucleotide sequence setforth in SEQ ID NO:1 or a fragment thereof; and isolating said DNAsequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genornicDNA to isolate full-length cDNAs and genomic clones encoding tyrS and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the tyrS gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the tyrS gene may be isolated byscreening using the DNA sequence provided in SEQ ID NO: 1 to synthesizean oligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genotnic DNA or mRNA to determine which members ofthe library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the tyrS polynucleotides ofthe invention for use as diagnostic reagents. Detection of tyrS in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also"individual(s)"), particularly mammals, and especially humans, infectedwith an organism comprising the tyrS gene may be detected at the nucleicacid level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genornic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled tyrS polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g., Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natt.Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding tyrS can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 2.

                  TABLE 2                                                         ______________________________________                                        Primers for amplification of tyrS polynucleotides                             SEQ ID NO   PRIMER SEQUENCE                                                   ______________________________________                                        5           5'-ATGCACATTTTTGATGAGCTAAAA-3'                                    6           5'-TTAGTAAGTCAATACAAAGTATTT-3'                                    ______________________________________                                    

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying tyrS DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, comprising determining from asample derived from an individual a increased level of expression ofpolynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. Increasedor decreased expression of tyrS polynucleotide can be measured using anyon of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of tyrS protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a tyrSprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-tyrS or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against tyrS- polypeptide may be employedto treat infections, particularly bacterial infections and especiallyotitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS, 1986:83,9551), encapsulation of DNA in various forms ofliposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS 1984:81,5849).

Antagonists and agonists--assays and molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of tyrSpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising tyrS polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a tyrS agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the tyrS polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of tyrS polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to chances in tyrS polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for tyrS antagonists is a competitive assaythat combines tyrS and a potential antagonist with tyrS-bindingmolecules, recombinant tyrS binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. tyrS can be labeled, such as byradioactivity or a colorimetric compound, such that the number of tyrSmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing tyrS-induced activities, thereby preventing the action of tyrSby excluding tyrS from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of tyrS.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock tyrS protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial tyrS proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with tyrS, or a fragment or variantthereof, adequate to produce antibody and/ or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Streptococcus pneumoniae infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector to direct expressionof tyrS, or a fragment or a variant thereof, for expressing tyrS, or afragment or a variant thereof in vivo in order to induce animmunological response, such as, to produce antibody and/ or T cellimmune response, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a tyrS or protein coded therefrom,wherein the composition comprises a recombinant tyrS or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid tyrS or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A tyrS polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzea, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352(1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and nonaqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain tyrSprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain Selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Streptococcuspneumoniae in E. coli. The sequencing data from two or more clonescontaining overlapping Streptococcus pneumoniae DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2

tyrS Characterization

The enzyme mediated incorporation of radiolabeled amino acid into tRNAmay be measured by the aminoacylation method which measures aminoacid-tRNA as trichloroacetic acid-precipitable radioactivity fromradiolabeled amino acid in the presence of tRNA and ATP (Hughes J,Mellows G and Soughton S, 1980, FEBS Letters, 122:322-324). Thusinhibitors of tyrosyl tRNA synthetase can be detected by a reduction inthe trichloroacetic acid precipitable radioactivity relative to thecontrol. Alternatively the tRNA synthetase catalysed partial PPi/ATPexchange reaction which measures the formation of radiolabeled ATP fromPPi can be used to detect tyrosyl tRNA synthetase inhibitors (Calender R& Berg P, 1966, Biochemistry, 5, 1681-1690).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 6                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1257 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Genomic DNA                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATGCACATTT TTGATGAGCT AAAAGAGCGT GGTTTGATAT TTCAAACGAC TG - #ATGAAGAA         60                                                                          - GCTTTGCGTA AAGCCCTAGA AGAAGGTCAA GTTTCTTATT ATACTGGCTA CG - #ATCCAACT        120                                                                          - GCTGACAGCC TTCACCTAGG CCACCTTGTC GCAATCTTGA CAAGTCGTCG CT - #TGCAACTA        180                                                                          - GCAGGTCACA AACCTTATGC GCTCGTTGGC GGTGCTACAG GTCTCATCGG AG - #ATCCGTCC        240                                                                          - TTCAAAGATG CTGAACGTAG TCTCCAAACA AAAGACACAG TAGATGGCTG GG - #TCAAGTCT        300                                                                          - ATCCAAGGAC AACTTTCTCG TTTTCTTGAC TTTGAAAATG GCGAAAACAA GG - #CTGTCATG        360                                                                          - GTCAACAACT ACGACTGGTT TGGCAGCATC AGCTTCATTG ACTTCCTCCG TG - #ATATTGGA        420                                                                          - AAATACTTCA CGGTCAACTA CATGATGAGT AAGGAATCTG TTAAAAAACG GA - #TCGAAACA        480                                                                          - GGAATTTCTT ACACTGAGTT CGCTTACCAA ATCATGCAAG GGTACGACTT CT - #TCGTCCTT        540                                                                          - AACCAAGACC ATAATGTCAC TCTTCAAATC GGTGGTTCTG ACCAGTGGGG AA - #ATATGACA        600                                                                          - GCTGGTACCG AATTGCTTCG TCGTAAGGCG GACAAGACTG GTCACGTTAT CA - #CTGTTCCA        660                                                                          - CTAATCACAG ATGCAACTGG TAAAAAATTT GGTAAATCAG AAGGAAATGC CG - #TCTGGCTC        720                                                                          - AATCCCGAAA AGACTTCTCC ATACGAAATG TACCAATTCT GGATGAACGT GA - #TGGACGCT        780                                                                          - GACGCTGTTC GCTTCTTGAA AATCTTTACT TTCTTGTCAC TTGATGAGAT TG - #AAGATATT        840                                                                          - CGTAAACAAT TTGAAGCAGC GCCACACGAA CGCTTGGCTC AAAAAGTCTT GG - #CTCGTGAA        900                                                                          - GTTGTTACAC TTGTTCACGG AGAAGAAGCC TACAAAGAAG CACTTAACAT CA - #CTGAGCAA        960                                                                          - CTCTTTGCAG GAAACATCAA AAACCTTTCT GTCAAAGAGC TCAAACAAGG AC - #TTCGTGGT       1020                                                                          - GTGCCAAACT ACCAAGTACA GGCAGACGAA AACAACAATA TCGTGGAACT GC - #TCGTCTCA       1080                                                                          - TCTGGTATAG TTAACTCAAA ACGCCAAGCC CGTGAAGACG TCCAAAACGG AG - #CCATCTAC       1140                                                                          - GTAAACGGCG ACCGCATCCA AGACCTTGAC TATGTCTTGA GTGACGCTGA TA - #AGTTAGAA       1200                                                                          - AATGAACTGA CTGTTATCCG TCGTGGGAAG AAAAAATACT TTGTATTGAC TT - #ACTAA          1257                                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 418 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met His Ile Phe Asp Glu Leu Lys Glu Arg Gl - #y Leu Ile Phe Gln Thr         #                15                                                           - Thr Asp Glu Glu Ala Leu Arg Lys Ala Leu Gl - #u Glu Gly Gln Val Ser         #            30                                                               - Tyr Tyr Thr Gly Tyr Asp Pro Thr Ala Asp Se - #r Leu His Leu Gly His         #        45                                                                   - Leu Val Ala Ile Leu Thr Ser Arg Arg Leu Gl - #n Leu Ala Gly His Lys         #    60                                                                       - Pro Tyr Ala Leu Val Gly Gly Ala Thr Gly Le - #u Ile Gly Asp Pro Ser         #80                                                                           - Phe Lys Asp Ala Glu Arg Ser Leu Gln Thr Ly - #s Asp Thr Val Asp Gly         #                95                                                           - Trp Val Lys Ser Ile Gln Gly Gln Leu Ser Ar - #g Phe Leu Asp Phe Glu         #           110                                                               - Asn Gly Glu Asn Lys Ala Val Met Val Asn As - #n Tyr Asp Trp Phe Gly         #       125                                                                   - Ser Ile Ser Phe Ile Asp Phe Leu Arg Asp Il - #e Gly Lys Tyr Phe Thr         #   140                                                                       - Val Asn Tyr Met Met Ser Lys Glu Ser Val Ly - #s Lys Arg Ile Glu Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Ile Ser Tyr Thr Glu Phe Ala Tyr Gln Il - #e Met Gln Gly Tyr Asp         #               175                                                           - Phe Phe Val Leu Asn Gln Asp His Asn Val Th - #r Leu Gln Ile Gly Gly         #           190                                                               - Ser Asp Gln Trp Gly Asn Met Thr Ala Gly Th - #r Glu Leu Leu Arg Arg         #       205                                                                   - Lys Ala Asp Lys Thr Gly His Val Ile Thr Va - #l Pro Leu Ile Thr Asp         #   220                                                                       - Ala Thr Gly Lys Lys Phe Gly Lys Ser Glu Gl - #y Asn Ala Val Trp Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Pro Glu Lys Thr Ser Pro Tyr Glu Met Ty - #r Gln Phe Trp Met Asn         #               255                                                           - Val Met Asp Ala Asp Ala Val Arg Phe Leu Ly - #s Ile Phe Thr Phe Leu         #           270                                                               - Ser Leu Asp Glu Ile Glu Asp Ile Arg Lys Gl - #n Phe Glu Ala Ala Pro         #       285                                                                   - His Glu Arg Leu Ala Gln Lys Val Leu Ala Ar - #g Glu Val Val Thr Leu         #   300                                                                       - Val His Gly Glu Glu Ala Tyr Lys Glu Ala Le - #u Asn Ile Thr Glu Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Phe Ala Gly Asn Ile Lys Asn Leu Ser Va - #l Lys Glu Leu Lys Gln         #               335                                                           - Gly Leu Arg Gly Val Pro Asn Tyr Gln Val Gl - #n Ala Asp Glu Asn Asn         #           350                                                               - Asn Ile Val Glu Leu Leu Val Ser Ser Gly Il - #e Val Asn Ser Lys Arg         #       365                                                                   - Gln Ala Arg Glu Asp Val Gln Asn Gly Ala Il - #e Tyr Val Asn Gly Asp         #   380                                                                       - Arg Ile Gln Asp Leu Asp Tyr Val Leu Ser As - #p Ala Asp Lys Leu Glu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asn Glu Leu Thr Val Ile Arg Arg Gly Lys Ly - #s Lys Tyr Phe Val Leu         #               415                                                           - Thr Tyr                                                                     - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 544 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Genomic DNA                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - AAGAGCTTGC GTAAAGCCCT AGAAGAAGGT CAAGTTTCTT ATTATACTGG CT - #ACGATCCA         60                                                                          - ACTGCTGACA GCCTTCACCT AGGCCACCTT GTCGCAATCT TGACAAGTCG TC - #GCTTGCAA        120                                                                          - CTAGCAGGTC ACAAACCTTA TGCGCTCGTT GGCGGTGCTA CAGGTCTCAT CG - #GAGATCCG        180                                                                          - TCCTTCAAAG ATGCTGAACG TAGTCTCCAA ACAAAAGACA CAGTAGATGG CT - #GGGTCAAG        240                                                                          - TCTATCCAAG GACAACTTTC TCGTTTTCTT GACTTTGAAA ATGGCGAAAA CA - #AGGCTGTC        300                                                                          - ATGGTCAACA ACTACGACTG GTTTGGCAGC ATCAGCTTCA TTGACTTCCT CC - #GTGATATT        360                                                                          - GGAAAATACT TCACGGTCAA CTACATGATG AGTAAGGAAT CTGTTAAAAA AC - #GGATCGAA        420                                                                          - ACAGGAATTT CTTACACTGA GTTCGCTTAC CAAATCATGC AAGGGTACGA CT - #TCTTCGTC        480                                                                          - CTTAACCAAG ACCATAATGT CACTCTTCAA ATCGGTGGTT CTGACCAGTG GG - #GAAATATG        540                                                                          #            544                                                              - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 181 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Lys Ser Leu Arg Lys Ala Leu Glu Glu Gly Gl - #n Val Ser Tyr Tyr Thr         #                15                                                           - Gly Tyr Asp Pro Thr Ala Asp Ser Leu His Le - #u Gly His Leu Val Ala         #            30                                                               - Ile Leu Thr Ser Arg Arg Leu Gln Leu Ala Gl - #y His Lys Pro Tyr Ala         #        45                                                                   - Leu Val Gly Gly Ala Thr Gly Leu Ile Gly As - #p Pro Ser Phe Lys Asp         #    60                                                                       - Ala Glu Arg Ser Leu Gln Thr Lys Asp Thr Va - #l Asp Gly Trp Val Lys         #80                                                                           - Ser Ile Gln Gly Gln Leu Ser Arg Phe Leu As - #p Phe Glu Asn Gly Glu         #                95                                                           - Asn Lys Ala Val Met Val Asn Asn Tyr Asp Tr - #p Phe Gly Ser Ile Ser         #           110                                                               - Phe Ile Asp Phe Leu Arg Asp Ile Gly Lys Ty - #r Phe Thr Val Asn Tyr         #       125                                                                   - Met Met Ser Lys Glu Ser Val Lys Lys Arg Il - #e Glu Thr Gly Ile Ser         #   140                                                                       - Tyr Thr Glu Phe Ala Tyr Gln Ile Met Gln Gl - #y Tyr Asp Phe Phe Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Asn Gln Asp His Asn Val Thr Leu Gln Il - #e Gly Gly Ser Asp Gln         #               175                                                           - Trp Gly Asn Met Thr                                                                     180                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #                24AGCT AAAA                                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Genomic DNA                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #                24AAGT ATTT                                                  __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide encoding a polypeptide comprising amino acids 1 to 418 ofSEQ ID NO:2.
 2. An isolated polynucleotide consisting of nucleotides 1to 1254 set forth in SEQ ID NO:1.
 3. An isolated polynucleotidecomprising nucleotides 1 to 1254 set forth in SEQ ID NO:1.
 4. Anisolated polynucleotide comprising nucleotides 1 to 1257 set forth inSEQ ID NO:1.
 5. An isolated polynucleotide of the formula (SEQ ID NO:1):

    __________________________________________________________________________    X-(R.sub.1).sub.n                                                             1      ATGCACATTT TTGATGAGCT AAAAGAGCGT GGTTTGATAT TTCAAACGAC                    51  TGATGAAGAA GCTTTGCGTA AAGCCCTAGA AGAAGGTCAA GTTTCTTATT                    101 ATACTGGCTA CGATCCAACT GCTGACAGCC TTCACCTAGG CCACCTTGTC                    151 GCAATCTTGA CAAGTCGTCG CTTGCAACTA GCAGGTCACA AACCTTATGC                    201 GCTCGTTGGC GGTGCTACAG GTCTCATCGG AGATCCGTCC TTCAAAGATG                    251 CTGAACGTAG TCTCCAAACA AAAGACACAG TAGATGGCTG GGTCAAGTCT                    301 ATCCAAGGAC AACTTTCTCG TTTTCTTGAC TTTGAAAATG GCGAAAACAA                    351 GGCTGTCATG GTCAACAACT ACGACTGGTT TGGCAGCATC AGCTTCATTG                    401 ACTTCCTCCG TGATATTGGA AAATACTTCA CGGTCAACTA CATGATGAGT                    451 AAGGAATCTG TTAAAAAACG GATCGAAACA GGAATTTCTT ACACTGAGTT                    501 CGCTTACCAA ATCATGCAAG GGTACGACTT CTTCGTCCTT AACCAAGACC                    551 ATAATGTCAC TCTTCAAATC GGTGGTTCTG ACCAGTGGGG AAATATGACA                    601 GCTGGTACCG AATTGCTTCG TCGTAAGGCG GACAAGACTG GTCACGTTAT                    651 CACTGTTCCA CTAATCACAG ATGCAACTGG TAAAAAATTT GGTAAATCAG                    701 AAGGAAATGC CGTCTGGCTC AATCCCGAAA AGACTTCTCC ATACGAAATG                    751 TACCAATTCT GGATGAACGT GATGGACGCT GACGCTGTTC GCTTCTTGAA                    801 AATCTTTACT TTCTTGTCAC TTGATGAGAT TGAAGATATT CGTAAACAAT                    851 TTGAAGCAGC GCCACACGAA CGCTTGGCTC AAAAAGTCTT GGCTCGTGAA                    901 GTTGTTACAC TTGTTCACGG AGAAGAAGCC TACAAAGAAG CACTTAACAT                    951 CACTGAGCAA CTCTTTGCAG GAAACATCAA AAACCTTTCT GTCAAAGAGC                    1001                                                                              TCAAACAAGG ACTTCGTGGT GTGCCAAACT ACCAAGTACA GGCAGACGAA                    1051                                                                              AACAACAATA TCGTGGAACT GCTCGTCTCA TCTGGTATAG TTAACTCAAA                    1101                                                                              ACGCCAAGCC CGTGAAGACG TCCAAAACGG AGCCATCTAC GTAAACGGCG                    1151                                                                              ACCGCATCCA AGACCTTGAC TATGTCTTGA GTGACGCTGA TAAGTTAGAA                    1201                                                                              AATGAACTGA CTGTTATCCG TCGTGGGAAG AAAAAATACT TTGTATTGAC                    1251                                                                              TTACTAA-(R.sub.2).sub.n -Y                                             __________________________________________________________________________

wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' endof the molecule, Y is hydrogen or a metal, R₁ and R₂ is any nucleic acidresidue, and n is an integer between 1 and
 1000. 6. An isolatedpolynucleotide encoding a polypeptide of the formula (SEQ ID NO:2):

    __________________________________________________________________________    X-(R.sub.1).sub.n                                                             1      MHIFDELKER GLIFQTTDEE ALRKALEEGQ VSYYTGYDPT ADSLHLGHLV                    51  AILTSRRLQL AGHKPYALVG GATGLIGDPS FKDAERSLQT KDTVDGWVKS                    101 IQGQLSRFLD FENGENKAVM VNNYDWFGSI SFIDFLRDIG KYFTVNYMMS                    151 KESVKKRIET GISYTEFAYQ IMQGYDFFVL NQDHNVTLQI GGSDQWGNMT                    201 AGTELLRRKA DKTGHVITVP LITDATGKKF GKSEGNAVWL NPEKTSPYEM                    251 YQFWMNVMDA DAVRFLKIFT FLSLDEIEDI RKQFEAAPHE RLAQKVLARE                    301 VVTLVHGEEA YKEALNITEQ LFAGNIKNLS VKELKQGLRG VPNYQVQADE                    351 NNNIVELLVS SGIVNSKRQA REDVQNGAIY VNGDRIQDLD YVLSDADKLE                    401 NELTVIRRGK KKYFVLTY-(R.sub.2).sub.n -Y                                 __________________________________________________________________________

wherein, at the amino terminus, X is hydrogen, and at the carboxylterminus, Y is hydrogen or a metal, R₁ and R₂ is any amino acid residue,and n is an integer between 1 and
 1000. 7. An isolated polynucleotidecomprising a polynucleotide encoding the same mature polypeptideexpressed by a polynucleotide comprising SEQ ID NO:1 in deposited strainNCIMB 40794 or NCIMB
 40800. 8. An isolated polynucleotide comprising thepolynucleotide of SEQ ID NO:3.
 9. An isolated polynucleotide comprisinga polynucleotide encoding a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:4.
 10. An isolated polynucleotidecomprising a first polynucleotide, or the full complement of the entirelength of the first polynucleotide; wherein the first polynucleotide hasat least 70% identity relative to a reference polynucleotide; whereinthe reference polynucleotide encodes the amino acid sequence of SEQ IDNO: 2 or 4; and wherein % identity is calculated as [1-N_(n) /X_(n)]×100; wherein N_(n) is the number of nucleotides in the firstpolynucleotide that are substituted, deleted or inserted when comparedto the reference polynucleotide, which is X_(n) nucleotides in length.11. The isolated polynucleotide of claim 10, wherein the firstpolynucleotide has at least 80% identity relative to the referencepolynucleotide, and comprising either the first polynucleotide or thefull complement of entire length of the first polynucleotide.
 12. Theisolated polynucleotide of claim 10, wherein the first polynucleotidehas at least 90% identity relative to the reference polynucleotide, andcomprising either the first polynucleotide or the full complement ofentire length of the first polynucleotide.
 13. The isolatedpolynucleotide of claim 10, wherein the first polynucleotide has atleast 95% identity relative to the reference polynucleotide; wherein thereference polynucleotide encodes the amino acid sequence of SEQ ID NO:2;and comprising either the first polynucleotide or the full complement ofentire length of the first polynucleotide.
 14. The isolatedpolynucleotide of claim 13, wherein the isolated polynucleotide is DNA.15. The isolated polynucleotide of claim 13, wherein the isolatedpolynucleotide is RNA.
 16. A vector comprising the isolatedpolynucleotide of claim
 13. 17. An isolated host cell comprising thevector of claim
 16. 18. A process for producing a polypeptide,comprising the step of culturing the host cell of claim 17 underconditions suitable for production of the polypeptide, wherein the firstpolynucleotide encodes an amino acid sequence selected from the groupconsisting of(a) an amino acid sequence comprising SEQ ID NO:2; (b) anamino acid sequence identical to SEQ ID NO:2 except that, over theentire length corresponding to SEQ ID NO:2, the amino acid sequence hasa substitution, deletion or insertion of one amino acid; (c) an aminoacid sequence comprising a portion of SEQ ID NO:2 containing at least 50amino acids; and, (d) an amino acid sequence comprising a portion of SEQID NO:2 containing at least 30 amino acids.
 19. The isolatedpolynucleotide of claim 10, wherein the first polynucleotide has atleast 95% identity relative to the reference polynucleotide; wherein thereference polynucleotide encodes the amino acid sequence of SEQ ID NO:4;and comprising either the first polynucleotide or the full complement ofentire length of the first polynucleotide.
 20. The isolatedpolynucleotide of claim 19, wherein the isolated polynucleotide is DNA.21. The isolated polynucleotide of claim 19 wherein the isolatedpolynucleotide is RNA.
 22. A vector comprising the isolatedpolynucleotide of claim
 19. 23. An isolated host cell comprising thevector of claim
 22. 24. A process for producing a polypeptide,comprising the step of culturing the host cell of claim 23 underconditions suitable for production of the polypeptide, wherein the firstpolynucleotide encodes an amino acid sequence selected from the groupconsisting of(a) an amino acid sequence comprising SEQ ID NO:4; (b) anamino acid sequence identical to SEQ ID NO:4 except that, over theentire length corresponding to SEQ ID NO:4, the amino acid sequence hasa substitution, deletion or insertion of one amino acid; (c) an aminoacid sequence comprising a portion of SEQ ID NO:4 containing at least 50amino acids; and, (d) an amino acid sequence comprising a portion of SEQID NO:4 containing at least 30 amino acids.
 25. An isolatedpolynucleotide comprising a first polynucleotide, or the full complementof the entire length of the first polynucleotide, wherein the firstpolynucleotide has at least 70% identity relative to a referencepolynucleotide encoding the same mature polypeptide expressed by apolynucleotide comprising SEQ ID NO: 1 or 3 in deposited strain NCIMB40794 or NCIMB 40800; wherein % identity is calculated as [1-N_(n)/X_(n) ]×100; wherein N_(n) is the number of nucleotides in the firstpolynucleotide that are substituted, deleted or inserted when comparedto the reference polynucleotide, which is X_(n) nucleotides in length.26. The isolated polynucleotide of claim 25, wherein the firstpolynucleotide has at least 80% identity relative to the referencepolynucleotide, and comprising either the first polynucleotide or thefull complement of entire length of the first polynucleotide.
 27. Theisolated polynucleotide of claim 25, wherein the first polynucleotidehas at least 90% identity relative to the reference polynucleotide, andcomprising either the first polynucleotide or the full complement ofentire length of the first polynucleotide.
 28. The isolatedpolynucleotide of claim 25, wherein the first polynucleotide has atleast 95% identity relative to the reference polynucleotide; wherein thereference polynucleotide encodes the same mature polypeptide expressedby the gene comprising the polynucleotide sequence of SEQ ID NO:1; andcomprising either the first polynucleotide or the full complement ofentire length of the first polynucleotide.
 29. A vector comprising theisolated polynucleotide of claim
 28. 30. An isolated host cellcomprising the vector of claim
 29. 31. The isolated polynucleotide ofclaim 25, wherein the first polynucleotide has at least 95% identityrelative to the reference polynucleotide; wherein the referencepolynucleotide encodes the same mature polypeptide expressed by the genecomprising the polynucleotide sequence of SEQ ID NO:3; and comprisingeither the first polynucleotide or the full complement of entire lengthof the first polynucleotide.
 32. A vector comprising the isolatedpolynucleotide of claim
 31. 33. An isolated host cell comprising thevector of claim
 32. 34. An isolated polynucleotide comprising a firstpolynucleotide, or the full complement of the entire length of the firstpolynucleotide, wherein the first polynucleotide hybridizes to the fullcomplement of the entire length of a reference polynucleotide as setforth in SEQ ID NO:1, wherein the first polynucleotide has at least 95%identity relative to the reference polynucteotide; wherein % identity iscalculated as [1-N_(n) /X_(n) ]×100; wherein N_(n) is the number ofnucleotides in the reference polynucleotide that are substituted,deleted or inserted when compared to SEQ ID NO:1, which is X_(n)nucleotides in length; and wherein the hybridization conditions includeincubation at 42° C. in a solution comprising: 50% formamide, 5× SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5× Denhardt's solution, 10% dextran sulfate, and 20 micrograms/mldenatured, sheared salmon sperm DNA, followed by washing in 0.1× SSC atabout 65° C.
 35. The isolated polynucleotide of claim 34, wherein thefirst polynucleotide has at least 97% identity relative to the referencepolynucleotide, and comprising either the first polynucleotide or thefull complement of entire length of the first polynucleotide.
 36. Avector comprising the isolated polynucleotide of claim
 34. 37. Anisolated host comprising the vector of claim
 36. 38. A process forproducing a polypeptide, comprising the step of culturing the host cellof claim 37 under conditions suitable for production of the polypeptide,wherein the first polynucleotide encodes an amino acid sequence selectedfrom the group consisting of(a) an amino acid sequence comprising SEQ IDNO:2; (b) an amino acid sequence identical to SEQ ID NO:2 except that,over the entire length corresponding to SEQ ID NO:2, the amino acidsequence has a substitution, deletion or insertion of one amino acid;(c) an amino acid sequence comprising a portion of SEQ ID NO:2containing at least 50 amino acids; and, (d) an amino acid sequencecomprising a portion of SEQ ID NO:2 containing at least 30 amino acids.39. An isolated polynucleotide comprising a first polynucleotide, or thefull complement of the entire length of the first polynucleotide,wherein the first polynucleotide hybridizes to the full complement ofthe entire length of a reference polynucleotide as set forth in SEQ IDNO:3, wherein the first polynucleotide has at least 95% identityrelative to the reference polynucleotide; wherein % identity iscalculated as [1-N_(n) /X_(n) ]×100; wherein N_(n) is the number ofnucleotides in the reference polynucleotide that are substituted,deleted or inserted when compared to SEQ ID NO:3, which is X_(n)nucleotides in length; and wherein the hybridization conditions includeincubation at 42° C. in a solution comprising 50% formamide, 5× SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 micrograms/mldenatured, sheared salmon sperm DNA, followed by washing in 0.1× SSC atabout 65° C.
 40. The isolated polynucleotide of claim 39 wherein thefirst polynucleotide has at least 97% identity relative to the referencepolynucleotide, and comprising either the first polynucleotide or thefull complement of entire length of the first polynucleotide.
 41. Avector comprising the isolated polynucleotide of claim
 39. 42. Anisolated host cell comprising the vector of claim
 41. 43. A process forproducing a polypeptide, comprising the step of culturing the host cellof claim 42 under conditions suitable for production of the polypeptide,wherein the first polynucleotide encodes an amino acid sequence selectedfrom the group consisting of(a) an amino acid sequence comprising SEQ IDNO:4; (b) an amino acid sequence identical to SEQ ID NO:4 except that,over the entire length corresponding to SEQ ID NO:4, the amino acidsequence has a substitution, deletion or insertion of one amino acid;(c) an amino acid sequence comprising a portion of SEQ ID NO:4containing al least 50 amino acids; and, (d) an amino acid sequencecomprising a portion of SEQ ID NO:4 containing at least 30 amino acids.44. An isolated polynucleotide comprising a polynucleotide encoding thesame mature polypeptide expressed by a polynucleotide comprising SEQ IDNO: 3 in deposited strain NCIMB 40794 or NCIMB
 40800. 45. A method forproducing antibodies in a mammal comprising: delivering to a tissue ofthe mammal a nucleic acid vector to direct expression in vivo apolypeptide from the isolated polynucleotide of claim 13, wherein thefirst polynucleotide encodes an amino acid sequence selected from thegroup consisting of(a) an amino acid sequence comprising SEQ ID NO:2;(b) an amino acid sequence identical to SEQ ID NO:2 except that, overthe entire length corresponding to SEQ ID NO:2, the amino acid sequencehas a substitution, deletion or insertion of one amino acid; (c) anamino acid sequence comprising a portion of SEQ ID NO:2 containing atleast 50 amino acids; and, (d) an amino acid sequence comprising aportion of SEQ ID NO:2 containing at least 30 amino acids;wherein thepolypeptide is effective to induce an immunological response to thepolypeptide of SEQ ID NO:2; and, wherein the polypeptide is expressed invivo and induces an immunological response to produce antibodies to thepolypeptide of SEQ ID NO:2.
 46. A method for producing antibodies in amammal comprising: delivering to a tissue of the mammal a nucleic acidvector to direct expression in vivo a polypeptide from the isolatedpolynucleotide of claim 19, wherein the first polynucleotide encodes anamino acid sequence selected from the group consisting of(a) an aminoacid sequence comprising SEQ ID NO:4; (b) an amino acid sequenceidentical to SEQ ID NO:4 except that, over the entire lengthcorresponding to SEQ ID NO:4, the amino acid sequence has asubstitution, deletion or insertion of one amino acid; (c) an amino acidsequence comprising a portion of SEQ ID NO:4 containing at least 50amino acids; and, (d) an amino acid sequence comprising a portion of SEQID NO:4 containing at least 30 amino acids;wherein the polypeptide iseffective to induce an immunological response to the polypeptide of SEQID NO:4; and, wherein the polypeptide is expressed in vivo and inducesan immunological response to produce antibodies to the polypeptide ofSEQ ID NO:4.